A new technology to determine base sequence of DNA or RNA is developed.
At present, in a normally used method of using electrophoresis, a cDNA fragment sample synthesized in advance through a reverse transcription reaction of a DNA fragment or RNA sample for sequencing is prepared, electrophoresis is performed after a dideoxy reaction by the well-known Sanger method is performed, and a molecular weight separation/expansion pattern is measured and analyzed.
On the other hand, in recent years, a method is proposed in which multiple DNA fragments as samples are fixed on a substrate, and sequence information of the multiple fragments is determined in parallel.
In Non Patent Literature 1, fine particles are used as vehicles for carrying DNA fragments, and PCR is performed on the fine particles. Thereafter, the fine particles carrying the PCR-amplified DNA fragments are placed on a plate provided with multiple holes each having a hole diameter corresponding to the size of the fine particle, and are read by a pyrosequence method.
In Non Patent Literature 2, fine particles are used as vehicles for carrying DNA fragments, and PCR is performed on the fine particles. Thereafter, the fine particles are dispersed on a glass substrate and are fixed, an enzyme reaction (ligation) is performed on the glass substrate, substrates with fluorescent dyes are captured and fluorescent detection is performed, so that sequence information of the respective fragments is obtained.
In Non Patent Literature 3, multiple DNA probes having the same sequence are fixed on a smooth substrate. Besides, after a DNA sample is cut, a DNA probe sequence and an adapter sequence of a complementary strand are added to ends of each of the DNA sample fragments. These are hybridised on the substrate, so that the sample DNA fragments are randomly fixed one molecule by one molecule on the substrate. In this case, a DNA extension reaction is performed on the substrate, and after substrates with fluorescent dyes are captured, washing of unreacted substrates and fluorescent detection are performed, and the sequence information of the sample DNA is obtained.
As described above, the method is developed in which multiple nucleic acid fragment samples are fixed on the smooth substrate, so that the sequence information of the multiple fragments is determined in parallel, and is being put to practical use.
As a nucleic acid analysis reaction cell used in these systems, it is desirable that a detection area where a sample DNA or a fine particle carrying a sample DNA is firmly fixed is wider. Besides, in order to reduce the amount of sample DNA necessary for the sequence reaction and the amount of reaction reagent, it is desirable that the volume in the flow cell is less. Further, in order to reduce a dead volume outside the detection area, an inflow port of a reagent is required to be narrowed.
Besides, a chemical reaction in these methods generally includes many steps using different reagents, and it is necessary to supply solutions containing the different reagents in the respective steps. A reagent used in a former step often becomes a reaction inhibition factor or a cause of erroneous detection in the next step, and it is necessary to perform a washing step between the steps and to completely remove the reagent used in the former step.
Patent Literature 1 discloses that a columnar member for reducing a cross section is provided in a portion where the width of a biochemical reaction cassette is wide.